Alessandra d'Azzo è titolare della Cattedra di Genetica e Terapia Genica di Jewelers for Children (JFC). Questo lavoro è stato sostenuto in parte dalle sovvenzioni NIH R01GM104981, RO1DK095169 e CA021764, dalla Fondazione Assisi di Memphis e dall'American Lebanese Syrian Associated Charities.

Tutte le procedure nei topi sono state eseguite secondo i protocolli sugli animali approvati dal St. Jude Children's Research Hospital Institutional Animal Care and Use Committee e dalle linee guida del National Institutes of Health.
1. Preparazione delle soluzioni e dei terreni
<ol><li>Preparare la soluzione di digestione mescolando 15,4 mL di PBS con 2,5 mL di 20 mg/mL di collagenasi P (concentrazione finale di 5 mg/mL), 2 mL di dispasi II 11 U/mL (concentrazione finale di 1,2 U/mL) e 100 μL di CaCl2 1,0 M (concentrazione finale di 5 mM).</li>
	<li>Preparare 500 mL di terreno fibroblastico primario (DMEM completo) mescolando 440 mL di DMEM con 50 mL di FBS (10%), 5,0 mL di penna/streptococco (100 U/mL e 100 μg/mL, rispettivamente) e 5,0 mL di integratore di glutammina (2 mM). Sterilizzare il mezzo con un filtro sottovuoto con pori da 0,22 μm.</li>
	<li>Preparare il siero privo di esosomi mediante ultracentrifugazione notturna di FBS in provette di polipropilene da 38,5 mL a 100.000 x g a 4 °C e trasferire il surnatante in una nuova provetta.</li>
	<li>Preparare 500 mL di terreno privo di esosomi mescolando 440 mL di DMEM con 50 mL di siero privo di esosomi (10%), 5 mL di penna/streptococco (100 U/mL e 100 μg/mL) e 5 mL di integratore di glutammina (2 mM). Sterilizzare il mezzo con un filtro sottovuoto con pori da 0,22 μm.</li>
	<li>Preparare Tris-HCl 0,5 M (pH 7,4) sciogliendo 6,057 g di Tris base in 90 mL di dH 2 O e regolando il pH a7,4 con HCl. Regolare il volume a 100 mL con dH2O.</li>
	<li>Preparare 10 mM di soluzione di Tris-HCl (pH 7,4)/1 mM di Mg(Ac)2 (soluzione di lavoro di saccarosio) mescolando 97,9 mL di dH 2 O con 2mL di Tris-HCl 0,5 M (pH = 7,4) e 100 μL di 1 M Mg(Ac)2. Aggiungere gli inibitori della proteasi alla soluzione appena prima dell'uso e mantenere la soluzione in ghiaccio.</li>
	<li>Preparare le soluzioni a gradiente di densità del saccarosio su ghiaccio secondo la Tabella 1. Queste soluzioni sono sufficienti per preparare due tubi a gradiente di saccarosio.</li>
	<li>Preparare il TCA al 100% aggiungendo 40 mL di dH2O a 100 g di TCA in polvere e mescolare fino a completo scioglimento. Regolare il volume finale con dH2O a 100 mL.</li>
	<li>Preparare l'etanolo all'80% mescolando 20 mL di dH2O con 80 mL di etanolo al 100%.</li>
	<li>Preparare il tampone di esecuzione Western blot mescolando 1,8 L di dH2O con 200 mL di tampone di esecuzione 10x.</li>
	<li>Preparare il tampone di trasferimento western blot mescolando 1,4 L di dH2O con 200 mL di tampone di trasferimento 10x e 400 ml di metanolo. Preraffreddare il tampone di trasferimento a 4 °C.</li>
	<li>Preparare 20 soluzione salina tamponata con Tris (TBS) sciogliendo 122 g di base Tris e 180 g di cloruro di sodio in 850 mL di dH2O (pH 8,0). Regolare il volume a 1 L con dH2O.</li>
	<li>Preparare il tampone bloccante sciogliendo 5 g di latte in polvere scremato con 1 mL di Tween-20 al 10%, 5 ml di TBS 20x e 94 ml di dH2O.</li>
	<li>Preparare il tampone anticorpale sciogliendo 3 g di BSA con 1 mL di Tween-20 al 10%, 5 mL di TBS 20x e 94 mL di dH2O.</li>
	<li>Preparare il tampone di lavaggio mescolando 100 mL di 20x TBS e 20 mL di 10% Tween-20 (10x) con 1,88 L di dH2O.</li>
	<li>Preparare la soluzione di sviluppo mescolando 1 volume di luminol/enhance con 1 volume di tampone di perossido stabile.</li>
</ol>2. Dissezione del muscolo del topo gastrocnemio (GA)15,16
<ol><li>Preparare provette da 50 mL contenenti 10 mL di PBS su ghiaccio.</li>
	<li>Eutanasia dei topi in una camera di CO2 seguita da lussazione cervicale.</li>
	<li>Rimuovere il muscolo gastrocnemio da entrambe le gambe e trasferirle nel tubo con PBS su ghiaccio. NOTA: Rimuovere il muscolo soleo dal muscolo GA prima di trasferire il muscolo GA al PBS.</li>
</ol>3. Isolamento e coltura di fibroblasti primari di topo
<ol><li>Pesare i muscoli e trasferirli in un piatto da 10 cm sotto una cappa di biosicurezza. Tritare i muscoli con il bisturi fino a quando non diventa una pasta fine.</li>
	<li>Trasferire la pasta muscolare finemente tritata in una provetta da 50 mL e aggiungere 3,5 volte il volume/mg di tessuto di soluzione digestiva nella provetta e incubare per 45 minuti a 37 °C. Mescolare accuratamente la sospensione con una pipetta da 5 ml ogni 10 minuti (questo aiuterà nella completa dissociazione). NOTA: In alternativa, è possibile utilizzare un dissociatore tissutale per questo passaggio.</li>
	<li>Aggiungere 20 mL di DMEM completo alla sospensione cellulare per inattivare la soluzione di digestione. Trasferire in un colino per celle di nylon da 70 μm posto su una provetta da 50 ml. Raccogliere il flusso passante e lavare il filtro cellulare con altri 5 ml di DMEM completo.</li>
	<li>Centrifugare la sospensione cellulare a 300 x g a temperatura ambiente (RT) per 10 minuti e rimuovere con cautela il surnatante.</li>
	<li>Risospendere il pellet in 10 mL di DMEM completo e seminare le cellule in un piatto di 10 cm.</li>
	<li>Coltivare le cellule a 37 °C, 5% CO 2, 3% O2 (passaggio 0 o P0). NOTA: 1) Queste colture devono essere mantenute a un basso livello di ossigeno per garantire condizioni simili a quelle fisiologiche (il livello di O 2 nel muscolo scheletrico è di circa il2,5%)17. 2) Il numero di passaggio (ad esempio, P0) di una coltura cellulare è una registrazione del numero di volte in cui la coltura è stata sottocoltivata. 3) I fibroblasti primari a P0 vengono coltivati di routine fino al 100% di confluenza più 1 giorno (e solo per P0). Questo per eliminare altri tipi di cellule e garantire che le cellule presenti nella coltura siano solo fibroblasti, impoveriti di cellule miogeniche in base all'esame al microscopio. Il muscolo scheletrico di un animale adulto a 2 mesi di età contiene circa il 2% di progenitori miogenici4. Inoltre, i progenitori miogenici di solito non si attaccano alle piastre non rivestite; pertanto, vengono persi durante la subcoltura18,19,20. Le cellule miogeniche richiedono un terreno diverso (F10) integrato con il 20% di FBS e fattore di crescita dei fibroblasti basici (bFGF)18. Nel DMEM completo, i fibroblasti medi hanno un tasso di proliferazione più elevato rispetto alle cellule miogeniche, il che si traduce nell'eliminazione di queste cellule contaminanti prima del primo passaggio.</li>
	<li>Risciacquare le celle P0 con PBS quando sono confluenti al 100% più 1 giorno. Aggiungere 1 mL di soluzione di tripsinizzazione e incubare a 37 °C, 5% CO 2, 3% O2 per staccare le cellule. Interrompere l'attività enzimatica utilizzando 10 mL di DMEM completo.</li>
	<li>Centrifugare le cellule a 300 x g per 10 minuti a RT e risospendere il pellet in 20 mL di DMEM completo e seminare in un piatto di 15 cm (passaggio 1 o P1). Le cellule vengono cresciute fino all'80%-90% di confluenza e possono essere espanse fino al passaggio 3. NOTA: A seconda degli esperimenti a valle, è possibile utilizzare il passaggio 1, il passaggio 2 (P2) o il passaggio 3 (P3).</li>
</ol>4. Semina delle cellule e raccolta del terreno condizionato
<ol><li>Lavare i fibroblasti (P1, P2 o P3) con 10 mL di PBS, aggiungere 2,5 mL di soluzione di tripsinizzazione alle cellule e incubare a 37 °C, 5% CO 2, 3% O2 . Interrompere l'attività enzimatica utilizzando 10 mL di DMEM completo. NOTA: Dopo il passaggio 4, le cellule primarie vengono scartate e non devono essere utilizzate per ulteriori esperimenti, poiché cambiano caratteristiche.</li>
	<li>Raccogliere le cellule e centrifugare a 300 x g a RT per 10 min.</li>
	<li>Risospendere il pellet cellulare in 10 mL di terreno privo di esosomi e contare le cellule.</li>
	<li>Seminare le cellule a 1,5–2,0 x 106 cellule per piatto (15 cm) e incubare a 37 °C, 5% CO 2, 3% O2 .</li>
	<li>Raccogliere il terreno condizionato tra 16 e 24 ore in provette da 50 mL su ghiaccio. NOTA: Se le cellule non sono confluenti all'80%, è possibile aggiungere nuovamente terreno libero esosomiale fresco e raccoglierlo dopo un ulteriore periodo di 16-24 ore. Le due collezioni possono essere combinate per ulteriori lavorazioni.</li>
</ol>5. Purificazione degli esosomi mediante differenziale e ultracentrifugazione
NOTA: Tutti i passaggi vengono eseguiti a 4 °C o su ghiaccio. Bilanciare il tubo con un tubo pieno d'acqua quando necessario.
<ol><li>Centrifugare il terreno condizionato a 300 x g per 10 minuti per la rimozione delle cellule vive e trasferire il surnatante in una nuova provetta da 50 mL.</li>
	<li>Rimuovere le cellule morte mediante centrifugazione a 2.000 x g per 10 minuti e trasferire il surnatante in una provetta di polipropilene da 38,5 mL.</li>
	<li>Centrifugare il surnatante a 10.000 x g per 30 minuti per la rimozione di organelli, corpi apoptotici e frammenti di membrana. Trasferire il surnatante in una provetta ultratrasparente da 38,5 mL.</li>
	<li>Ultracentrifugare il surnatante a 100.000 x g per 1,5 h per pellettare gli esosomi.</li>
	<li>Scartare con cura il surnatante, lasciando circa 1 mL di terreno condizionato, e lavare il pellet dell'esosoma in un volume totale di 30 mL di PBS ghiacciato.</li>
	<li>Centrifugare a 100.000 x g per 1,5 h ed eliminare con cura il surnatante mediante pipettaggio, facendo attenzione a non disturbare il pellet esosomiale.</li>
	<li>Risospendere il pellet in PBS ghiacciato (~25 μL per piatto da 15 cm) e misurare la concentrazione proteica utilizzando il kit del saggio delle proteine BCA e un lettore di micropiastre a 562 nm. NOTA: La concentrazione proteica è misurata mediante una diluizione 1:2 con Triton-X100 allo 0,1% in dH2O. La resa degli esosomi da un piatto da 15 cm (20 mL di terreno condizionato) di fibroblasti primari all'80% di confluenza è di ~3-4 μg.</li>
</ol>6. Caratterizzazione degli esosomi mediante gradiente di densità del saccarosio
<ol><li>Caricare il gradiente di saccarosio in una provetta ultratrasparente da 13 mL pipettando (dal basso verso l'alto) quanto segue: 1,5 mL di 2,0 M, 2,5 mL di 1,3 M, 2,5 mL di 1,16 M, 2,0 mL di 0,8 M e 2,0 mL di soluzioni di saccarosio 0,5 M.</li>
	<li>Mescolare 30-100 μg di esosomi con una soluzione di saccarosio 0,25 M e regolare il volume a 1 mL.</li>
	<li>Caricare con cautela gli esosomi sopra i gradienti e ultracentrifugare i campioni per 2,5 ore a 100.000 x g e 4 °C.</li>
	<li>Rimuovere le provette dall'ultracentrifuga e posizionarle sul ghiaccio e raccogliere 1,0 mL di frazioni partendo dalla parte superiore del gradiente. Trasferirli in provette da 1,7 mL su ghiaccio.</li>
	<li>Aggiungere 110 μL di TCA al 100% in ciascuna provetta, mescolare bene e incubare per 10 minuti a RT.</li>
	<li>Centrifugare per 10 minuti a 10.000 x g e RT ed eliminare con cura il surnatante.</li>
	<li>Risospendere il pellet in 500 μL di etanolo preraffreddato all'80% e lasciare lavare i campioni per 10 minuti a -20 °C.</li>
	<li>Centrifugare per 10 minuti a 10.000 x g e 4 °C ed eliminare il surnatante.</li>
	<li>Asciugare il pellet per 10-15 minuti a RT e risospendere il pellet in PBS per esperimenti in vitro. Misurare la concentrazione proteica utilizzando il kit del saggio delle proteine BCA o risospendere il pellet direttamente in 14,5 μL di dH2O, 5 μL di tampone Laemmli 4x e 0,5 μL di 1 M DTT per le analisi western blot.</li>
</ol>7. Rilevamento degli esosomi mediante analisi western blot
<ol><li>Miscelare 5-10 μg di esosomi del passaggio 5.7 con 5 μL di tampone Laemmli 4x e 0,5 μL di 1 M DTT, quindi regolare il volume finale a 20 μL con dH2O. Riscaldare tutti i campioni, compresi quelli del passaggio 6.9, per 5 minuti a 98 °C.</li>
	<li>Caricare i campioni in un gel TGX privo di coloranti al 10% e caricare le scale proteiche secondo le raccomandazioni del produttore. NOTA: Scegliere la percentuale di gel in base al peso molecolare della proteina di interesse.</li>
	<li>Far scorrere il gel a 100 V per ~1 ora nel tampone di funzionamento fino a quando il colorante di caricamento non si trova sul fondo del gel. NOTA: Il tempo di funzionamento può variare in base all'apparecchiatura utilizzata o al tipo e alla percentuale di gel.</li>
	<li>Immagina il gel. Le immagini dei gel privi di colorazione vengono utilizzate come controllo del carico per gli immunoblots.</li>
	<li>Trasferire il gel con una membrana in PVDF per 1,5 ore a 80 V o per una notte a 30 V a 4 °C.</li>
	<li>Bloccare le membrane nel tampone di blocco per 1 ora a RT.</li>
	<li>Incubare le membrane per la ricerca di anticorpi specifici (Tabella 2) in tampone anticorpale per una notte a 4 °C con agitazione.</li>
	<li>Lavare le membrane in tampone di lavaggio e incubare le membrane con gli anticorpi secondari appropriati (Tabella 2) per 1 ora a RT con agitazione.</li>
	<li>Lavare le membrane in un tampone di lavaggio e creare un'immagine delle membrane utilizzando una soluzione di sviluppo e un imager basato su telecamera. In alternativa, utilizzare una pellicola a raggi X per sviluppare le membrane.</li>
</ol>

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Un passo fondamentale per il successo dell'isolamento degli esosomi dai terreni di coltura, come delineato in questo protocollo, è la corretta costituzione e mantenimento di colture primarie di fibroblasti di topo dal muscolo scheletrico adulto. Queste colture devono essere mantenute a un basso livello di ossigeno per garantire condizioni simili a quelle fisiologiche (il livello di O2 nel muscolo scheletrico è ~2,5%)15. I fibroblasti primari cambiano caratteristiche se passati in colt...

Gli esosomi sono vescicole extracellulari eterogenee di dimensioni comprese tra 30 e 150 nm. Sono attori chiave affermati nei processi fisiologici e patologici, data la loro distribuzione ubiquitaria nei tessuti e negli organi 1,2. Gli esosomi trasportano un carico complesso di proteine, lipidi, tipi di DNA e tipi di RNA, che variano a seconda del tipo di cellule da cui derivano 1,2,3. Gli esosomi sono arricchiti in proteine che hanno funzioni diverse (ad esempio, le tetraspanine, tra cui CD9 e CD63) sono responsabili degli eventi di fusione. Ad esempio, le proteine da shock termico HSP70 e HSP90 sono coinvolte nel legame e nella presentazione dell'antigene. Inoltre, Alix, Tsg101 e flotillina partecipano alla biogenesi e al rilascio degli esosomi e sono ampiamente utilizzati come marcatori di queste nanovescicole 2,3,4.
Gli esosomi contengono anche una varietà di RNA (cioè microRNA, RNA lunghi non codificanti, RNA ribosomiali) che possono essere trasferiti alle cellule riceventi, dove influenzano la segnalazione a valle3. Essendo racchiusi da una singola unità di membrana, la bioattività degli esosomi dipende non solo dal carico di proteine e acidi nucleici, ma anche dai componenti lipidici della membrana limitante1. Le membrane esosomiali sono arricchite in fosfatidilserina, acido fosfatidico, colesterolo, sfingomielina, acido arachidonico e altri acidi grassi, che possono influenzare la stabilità degli esosomi e la topologia della membrana 2,3. Come risultato della disposizione del carico e dei lipidi, gli esosomi avviano vie di segnalazione nelle cellule riceventi e partecipano al mantenimento della normale fisiologia tissutale 1,2,4,5. In alcune condizioni patologiche (ad esempio, neurodegenerazione, fibrosi e cancro), è stato dimostrato che innescano e propagano stimoli patologici 4,6,7,8,9,10,11.
Grazie alla loro capacità di propagare i segnali a siti vicini o lontani, gli esosomi sono diventati preziosi biomarcatori per la diagnosi o la prognosi delle condizioni patologiche. Inoltre, gli esosomi sono stati utilizzati sperimentalmente come veicoli dei composti terapeutici 2,12. La potenziale applicazione di queste nanovescicole in clinica rende il metodo di isolamento sempre più importante per ottenere la massima resa, purezza e riproducibilità. Sono state sviluppate e implementate diverse tecniche per l'isolamento degli esosomi. Generalmente, gli esosomi possono essere isolati da terreni di coltura cellulare condizionati o fluidi corporei mediante centrifugazione differenziale, cromatografia ad esclusione dimensionale e cattura immunitaria (utilizzando kit disponibili in commercio). Ogni approccio presenta vantaggi e svantaggi unici che sono stati discussi in precedenza 1,2,13,14.
Il protocollo delineato si concentra su 1) l'isolamento e la coltura di fibroblasti primari dal muscolo gastrocnemio di topo adulto e 2) la purificazione e la caratterizzazione degli esosomi rilasciati nel terreno di coltura da queste cellule. Attualmente manca un protocollo consolidato per l'isolamento degli esosomi dai fibroblasti primari per gli studi funzionali. I fibroblasti primari non secernono grandi quantità di esosomi, rendendo difficile il processo di isolamento e purificazione. Questo protocollo descrive la purificazione di grandi quantità di esosomi puri da grandi volumi di coltura, mantenendone l'integrità morfologica e l'attività funzionale. Gli esosomi purificati ottenuti da un terreno condizionato sono stati utilizzati con successo in esperimenti di captazione in vitro per indurre specifiche vie di segnalazione nelle cellule riceventi. Sono stati utilizzati anche per analisi proteomiche comparative di carichi esosomiali da più campioni biologici4.

<table>
	<tbody>
		<tr>
			<td>10 cm dishes</td>
			<td>Midwest Scientific, TPP</td>
			<td>TP93100</td>
			<td></td>
		</tr>
		<tr>
			<td>15 cm dishes</td>
			<td>Midwest Scientific</td>
			<td>TP93150</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA protein assay kit</td>
			<td>Thermo Fisher Scientific, Pierce</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin Fraction V</td>
			<td>Roche</td>
			<td>10735094001</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C1016-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5430R with rotors FA-35-6-30/ FA-45-48-11</td>
			<td>Eppendorf</td>
			<td>022620659/5427754008</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemidoc MP imaging system</td>
			<td>BioRad</td>
			<td>12003154</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase P</td>
			<td>Sigma, Roche</td>
			<td>11 213 857 001</td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>cOmplete protease inhibitor cocktail</td>
			<td>Millipore/Sigma, Roche</td>
			<td>11697498001</td>
			<td></td>
		</tr>
		<tr>
			<td>Criterion Blotter with plate electrodes</td>
			<td>BioRad</td>
			<td>1704070</td>
			<td></td>
		</tr>
		<tr>
			<td>Criterion TGX stain-free protein gel</td>
			<td>BioRad</td>
			<td>5678034</td>
			<td>10% 18-well, midi-gel</td>
		</tr>
		<tr>
			<td>Criterion vertical electrophoresis cell (midi)</td>
			<td>BioRad</td>
			<td>NC0165100</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Sigma, Roche</td>
			<td>04 942 078 001</td>
			<td>neutral protease, grade II</td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Sigma/Millipore, Roche</td>
			<td>10708984001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modification Eagles Medium</td>
			<td>Corning</td>
			<td>15-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>Pharmco by Greenfield Global</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 mL conical centrifuge tubes</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluostar Omega multi-mode microplate reader</td>
			<td>BMG Labtech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX supplement</td>
			<td>Thermo Fisher Scientific, Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Fisher Scientific</td>
			<td>A144S-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilon-P Transfer membranes</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Laemmli sample buffer (4x)</td>
			<td>BioRad</td>
			<td>1610747</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium acetate solution</td>
			<td>Sigma</td>
			<td>63052-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-fat dry milk</td>
			<td>LabScientific</td>
			<td>M-0842</td>
			<td></td>
		</tr>
		<tr>
			<td>O2/CO2 incubator</td>
			<td>Sanyo</td>
			<td>MC0-18M</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific, Gibco</td>
			<td>15140-122</td>
			<td>10,000 U/ml</td>
		</tr>
		<tr>
			<td>Premium microcentrifuge tubes</td>
			<td>Fisher Scientific, Midwest Scientific</td>
			<td>AVSC1510</td>
			<td>1.7 mL</td>
		</tr>
		<tr>
			<td>Protected disposable scalpels</td>
			<td>Fisher Scientific, Aspen Surgical Bard-Parker</td>
			<td>372610</td>
			<td></td>
		</tr>
		<tr>
			<td>Running buffer</td>
			<td>BioRad</td>
			<td>1610732</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific, Fisher Chemical</td>
			<td>S271-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup Quick release-GP sterile vacuum filtration system</td>
			<td>Millipore</td>
			<td>S2GPU05RE</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Sterile cell strainer (70 mm)</td>
			<td>Fisher Scientific, Fisher brand</td>
			<td>22-363-548</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific, Fisher Chemical</td>
			<td>S5-500</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperSignal west Femto</td>
			<td>Thermo Fisher Scientific</td>
			<td>34096</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin wall Polypropylene tubes</td>
			<td>Beckman Coulter</td>
			<td>326823</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer buffer</td>
			<td>BioRad</td>
			<td>16110734</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloroacetic Acid</td>
			<td>Sigma</td>
			<td>91228-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>BioRad</td>
			<td>1610719</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X100 solution</td>
			<td>Sigma</td>
			<td>93443-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme</td>
			<td>Thermo Fisher Scientific, Gibco</td>
			<td>12604-013</td>
			<td>No phenol red</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>BioRad</td>
			<td>#1610781</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-centrifuge Optima XPM</td>
			<td>Beckman Coulter</td>
			<td>A99842</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-clear tube (14x89 mm)</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-clear tubes (25x89 mm)</td>
			<td>Beckman Coulter</td>
			<td>344058</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath Isotemp 220</td>
			<td>Fisher Scientific</td>
			<td>FS220</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and characterization of exosomes from skeletal muscle fibroblasts

Gli esosomi sono piccole vescicole extracellulari rilasciate praticamente da tutte le cellule e secrete in tutti i fluidi biologici. Sono stati sviluppati molti metodi per l'isolamento di queste vescicole, tra cui l'ultracentrifugazione, l'ultrafiltrazione e la cromatografia ad esclusione dimensionale. Tuttavia, non tutti sono adatti per la purificazione e la caratterizzazione di esosomi su larga scala. Qui è delineato un protocollo per stabilire colture di fibroblasti primari isolati da muscoli scheletrici di topo adulto, seguito dalla purificazione e caratterizzazione degli esosomi dai terreni di coltura di queste cellule. Il metodo si basa sull'uso di fasi di centrifugazione sequenziale seguite da gradienti di densità del saccarosio. La purezza dei preparati esosomiali viene quindi convalidata mediante analisi western blot utilizzando una batteria di marcatori canonici (ad esempio, Alix, CD9 e CD81). Il protocollo descrive come isolare e concentrare gli esosomi bioattivi per la microscopia elettronica, la spettrometria di massa e gli esperimenti di uptake per studi funzionali. Può essere facilmente scalato verso l'alto o verso il basso e adattato per l'isolamento degli esosomi da diversi tipi di cellule, tessuti e fluidi biologici.

Questo protocollo è adatto per la purificazione di esosomi da grandi volumi di terreno condizionato in modo economico. La procedura è altamente riproducibile e coerente. La Figura 1 mostra l'immagine al microscopio elettronico a trasmissione (TEM) degli esosomi purificati dal terreno di coltura dei fibroblasti primari di topo. La Figura 2 mostra il pattern di espressione proteica dei marcatori esosomiali canonici e l'assenza di contaminanti proteici citosolici...

Watch this Scientific Journal Video about Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts at JoVE.com

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Questo protocollo illustra 1) l'isolamento e la coltura di fibroblasti primari dal muscolo gastrocnemio di topo adulto e 2) la purificazione e la caratterizzazione degli esosomi utilizzando un metodo di ultracentrifugazione differenziale combinato con gradienti di densità del saccarosio seguito da analisi western blot.

Isolamento e caratterizzazione di esosomi da fibroblasti muscolari scheletrici

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

isolation-characterization-exosomes-from-skeletal-muscle

Research

JoVE Journal

Biology

3.2K Views.  St. Jude Children's Research Hospital. Questo protocollo illustra 1) l'isolamento e la coltura di fibroblasti primari dal muscolo gastrocnemio di topo adulto e 2) la purificazione e la caratterizzazione degli esosomi utilizzando un metodo di ultracentrifugazione differenziale combinato con gradienti di densità del saccarosio seguito da analisi western blot.

Video: Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Adulte e embrionali muscolo scheletrico Microexplant Cultura e isolamento di cellule staminali del muscolo scheletrico

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Ricerca

Biologia

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

Isolamento dei mitocondri dal muscolo scheletrico

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Purificazione di progenitori dal muscolo scheletrico

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

Isolamento e caratterizzazione di RNA contenenti esosomi

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Isolamento e la coltura di miofibre individuali e delle loro cellule satelliti da adulto muscolo scheletrico

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Isolamento dei vasi sanguigni derivati ​​da Multipotent precursori del muscolo scheletrico umano

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

L&#39;isolamento di mitocondri da minime quantità di muscolo scheletrico del mouse per misurazioni High Throughput micropiastre respiratorie

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

L&#39;isolamento di mitocondri intatti dal muscolo scheletrico mediante centrifugazione differenziale per alta risoluzione respirometria Misure

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

Isolamento e caratterizzazione di fibroblasti cardiaci adulti e miofibroblasti

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

Isolamento dei cardiomiociti ventricolari umani dalle fette miocardici Vibratome-Cut

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...